Research Article

IUFS Journal of Biology IUFS J Biol 2008, 67(1):9-16

Research Article 9

Quantitative Microbiological Analysis of Biofilm Communities from the Surfaces of Different Cooling Tower Materials Ayten Kimiran-Erdem*, Nazmiye Ozlem Sanli-Yürüdü, Elif Özlem Arslan-Aydoğdu, Nihal Dogruoz, Zuhal Zeybek, Irfan Türetgen, Aysin Cotuk Istanbul University, Faculty of Science, Department of Biology, 34134, Vezneciler, Istanbul-Turkey

Abstract Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Since biofilm formation is influenced by the type of surface materials, in the current study it was aimed to compare copper, stainless steel, galvanized stainless steel, polyvinyl chloride, polyethylene, polypropylene, ceramic and glass surfaces for biofilm formation rate. In this study, both monthly collected water and biofilm samples were analyzed in terms of total coliforms, faecal coliforms, Pseudomonas, aerobic mesophilic heterotrophic bacteria (at 22 and 37°C) and amoebas. We found that plastic polymers, especially polyethylene and polypropylene, supported the lowest total aerobic mesophilic heterotrophic bacterial numbers. Although the protozoa (amoeba) could found on to all of the surfaces, Pseudomonas species could harbour none of them. It can be concluded that selection of the suitable pipe material could reduce waterborne disease and minimize the possibility of biofilm development associated with the operation of cooling tower systems. Keywords: Biofilm, cooling tower, surface material, bacteria, amoeba. *Corresponding author: Ayten Kimiran-Erdem (E-mail: [email protected])

Introduction Biofouling occurs in nearly every industrial water-based process, including water treatment and distribution, pulp-paper manufacturing, the operation of cooling towers and also in medical devices such as dental unit water lines, catheters or ventilators (Percival et al. 1998; Gagnon and Slawson 1999; Momba and Binda 2002). It is generally accepted that bacteria have a tendency to attach to surfaces and initiate biofilm formation (Xu et al. 1998). Biofilms are defined as functional consortia of microorganisms organized within their extracellular polymeric substances (EPS), which facilitate irreversible attachment of cells to the surface, inorganic precipitates derived from the bulk aqueous phase and/or corrosion products of the metal substratum (Beech and Gaylarde 1999). Biofilm layer provides mechanical stability (Stoodley et al. 2002), ideal growth conditions for

microorganisms and also protects its inhabitants from physico-chemical alterations occurring in the bulk water phase (Costerton 1999). In particular, protozoa may play an important role as predators of biofilm bacteria, however they can also act as protection for bacteria against exogenous influences i.e. disinfection. Biofilms lead to many undesired conditions in industry, such as decreased heat transfer in cooling towers, deterioration/corrosion of materials, increased resistance to antimicrobial compounds and growth in drinking water distribution systems (Lechevallier et al. 1988, Rogers et al. 1994b; Xu et al. 1998; Momba and Binda 2002; Schwartz et al. 2003; Sanlı-Yurudu et al. 2007). Furthermore, since biofilm can harbour infective microorganisms, detachment of cells from biofilms in water systems may result in the potential transmission of pathogens

10

A. Kimiran - Erdem et al. / IUFS Journal of Biology 2008, 67(1): 9-16

via contaminated food, drinking water, or aerosols and have potential to increase the risk of pathogen exposure to patients (Stoodley et al. 2002). The accumulation of microorganisms on the surfaces of pipe materials and the formation of biofilms depend on many factors prevailing in the water system, e.g. types of surface materials, pH, microbial occurrence in water, concentration and quality of nutrients, the microbial quality of intake water, and disinfectants, the presence of a disinfectant residual, water temperature, and hydraulics of the system (Niquette et al. 2000; Zacheus et al. 2000; Momba and Binda 2002). The characteristics of the surface material composing pipes may greatly influence the densities of bacteria fixed in a distribution system (Niquette et al. 2000; Momba and Makala 2004; Türetgen and Cotuk 2007). A wide range of cooling tower construction materials is used. During the study period, bulk water was used as the water source; materials such as copper (Cu), stainless steel (SS), galvanized stainless steel (GSS), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), ceramic (C) and glass (G) were preferred as test pipes for the study of biofilms. Since microbial biofilms cause problems both in medicine and industry (Xu et al. 1998), it would be beneficial to control biofilms. The main purpose of the current study was to compare the effect various pipe materials on biofilm formation and selection of the appropriate manufacturing material for water systems.

Material and Methods Model system and test coupons The experimental study was performed using a 100-liter polypropylene lab-scale cooling tower water system under constant hydraulic conditions, which correspond more with the situation in cooling tower installations. It is equipped with a recirculating pump in the basin and a heat source to facilitate evaporation. Cover lid has openings to ensure fresh air and daylight entry (Fig. 1). A supply of potable water was used to replenish water lost by evaporation and blowdown (partial draining). Throughout the experiment, the water temperature was kept constant at 29°C. Materials that are commonly used in construction of cooling tower systems were selected (Cu, SS, GSS, PVC, PE, PP, C, and G, as control due to its chemically inert characteristic). All the materials are in rigid form, certified and commercially available. The coupons (20 x 50 x 1 mm) were washed with detergent, rinsed with distilled water, immersed in 70% ethanol for 5 min and air dried before use (Bloomfield et al. 1993). Biofilms were allowed to develop for 5 months on coupons within the aqueous phase of the system. Coupons were inserted vertically into water basins. No chemicals (disinfectant, pH regulators or anti-scaling agents) were added to the system, to exclude their negative effects on microorganisms and biofilm formation.


11

Figure 1. Schematic diagram of model recirculating water system, arrows indicate the flow direction. P: pump, S: surfaces, H: heater, M: make-up water inlet, B: blowdown outlet

Microbiological Assessments of Biofilm and Water Samples Both water and biofilm samples were collected from the model system monthly. Samples were analyzed in terms of total coliforms (TC), faecal coliforms (FC), Pseudomonas, aerobic mesophilic heterotrophic bacteria (AMHB) (at 22 and 37°C) and amoeba.

Analysis of Water Samples Total and faecal coliforms were detected by membrane filtration on selective media, EndoNKS (Sartorious) for total coliforms and mFC agar (Sartorious) for faecal coliforms, according to the Standard Methods (APHA 1998). 100 ml water samples were filtered through membrane filter (0.45 µm pore-size) (Sartorius), and were then placed on the selective media. The mFC and Endo-NKS plates were incubated at 44o and 37°C, respectively, for 18–24 h. Then the characteristic blue colonies were counted as FC. TCs were detected by production of typical red colonies with a metallic surface sheen or atypical dark red colonies without sheen. For isolation of Pseudomonas species each water samples (100 ml) were filtered by cellulose membrane filter (0.45 µm) and these filters were placed on Pseudomonas CFC selective supplement added Pseudomonas base

agar (Oxoid). All plates were incubated at 27 ºC for 48 hours (Donnell et al. 2005). Plate count agar (PCA) (Oxoid) plates were inoculated with 0.1 ml bulk water samples for AMHB counts. After inoculation plates were incubated at 37 ºC and at 22 ºC for 24-48 hours. In the amoeba analysis, each of water samples was examined to wet-mount preparation under light microscope (40x) directly. Also, each of water samples (100 ml) were filtered by cellulose membrane filter (0.45 µm) and these filters were placed on nonnutrient agar (NNA) seeded with heat-killed Escherichia coli. All plates were incubated at 30ºC and examined microscopically for amoeba (trophozoite and cyst) everyday for 10 days (Jeong and Yu 2005). Each experiment was done in triplicate. At the end of the incubation periods, colonies on all plates were counted by a Colony Counter Device (åCOLyte Super Colony Counter, Synbiosis) and expressed as colony-forming units per ml (log cfu ml-1).

Analysis of Biofilm Samples Three coupons of each material were removed monthly from the basin, dip-rinsed in sterile phosphate buffer to remove unattached cells. Biofilms on surfaces were scraped by sterile scalpel, suspended in phosphate buffer


12

and vortexed for 60 s (Gagnon and Slawson 1999). Homogenated biofilm samples were diluted in 1/200 ratio, from diluted biofilm homogenates, 0.1 ml liquid was drawn and transferred to PCA, mFC, Endo-NKS and Pseudomonas CFC selective supplement added Pseudomonas base agar (Oxoid) for the isolation of AMHB (at 22°C and 37°C), FCs, TCs and Pseudomonas spp., respectively. All plates were incubated suitable temperatures and periods. At the end of the incubation periods, bacterial colonies on the plates were counted by a Colony Counter Device (åCOLyte Super Colony Counter, Synbiosis). Analyses were carried out in triplicate. At the end of incubation period, attached bacterial counts were expressed as log cfu cm-2, and calculated using the following equation: Attached viable count (cfu cm-2) = N x D /surface area of slides where: N = average number of colonies and D = dilution factor.

For the analysis of amoeba each biofilm suspensions were examined to wet-mount preparation under light microscope (40x) directly. Also, 10 µl of biofilm suspensions were taken and inoculated on non-nutrient agar (NNA) seeded with heat-killed Escherichia coli. All plates were incubated at 30ºC and examined microscopically for amoeba (trophozoite and cyst) everyday for 10 days (Jeong and Yu 2005).

Statistical Analysis Results were analyzed statistically by Student's t-test. Differences were considered significant when p